Hydrated autoclave pretreatment enhances tau immunoreactivity in formalin-fixed normal and Alzheimer's disease brain tissues

We developed a new immunohistochemical method by which normal tau antigenicity can be visualized in paraffin sections of formalin-fixed brain tissue. This method consists of autoclave pretreatment of sections immersed into distilled water (hydrated autoclaving) before incubation with anti-tau antibodies. In normal human brain, immunoreactive tau was detected in neuronal cell bodies and dendrites, axon fibers, astroglia, oligodendroglia and gray matter neuropil. In previous studies on normal tau distribution, different optimized fixations that effectively preserve tau antigenicity were used but none of these revealed all of these compartments together. Our method is therefore considered to be more sensitive for detecting normal tau immunoreactivity. In addition, hydrated autoclaving had an enhancing effect on the abnormally phosphorylated (modified) tau immunoreactivity in formalin-fixed brains. In hydrated autoclaving of sections from patients with Alzheimer's disease, neuropil threads, senile plaques, extracellular and intracellular tangles were enhanced in quantity and in staining intensity. Therefore, modified tau appears to accumulate more densely than expected from conventional immunohistochemistry. Immunoblot analysis showed that normal or modified tau immunoreactivity was totally or partially eliminated on formalin treatment and could be revisualized by hydrated autoclaving, an event presumably related to recovering of formalin-masked tau antigens through denaturation by hydrated autoclaving.     

Regions with abundant neurofibrillary pathology in human brain exhibit a selective reduction in levels of binding-competent tau and accumulation of abnormal tau-isoforms (A68 proteins).

Paired helical filaments, the dominant filamentous components of Alzheimer's disease (AD), neurofibrillary tangles (NFT), neuropil threads, and the dystrophic neurites associated with amyloid rich senile plaques, are composed of abnormally phosphorylated derivatives of tau known as A68 proteins. Indeed the inappropriate phosphorylation of Ser396, which is adjacent to the microtuble binding domain in tau, may contribute to the transformation of tau into A68 and prevent A68 from efficiently binding to microtubules. The reduced levels of normal soluble tau proteins in AD brains may be the consequence of a multi-step process whereby normal tau is converted into A68 and sequestered in paired helical filaments. To elucidate the events involved in this process, we compared the relative levels of binding-competent (BC) and binding-incompetent (BI) tau with the level of A68 in six different regions (hippocampus, fornix, frontal grey and white matter, and cerebellar grey and white matter) of fresh AD and control brains. When the AD brains were compared as a group with neurologically normal and diseased non-AD controls, quantitative immunoblot analysis demonstrated a selective reduction of BC tau in regions of the AD brains with abundant neurofibrillary lesions (NFTs, neuropil threads, and senile plaque neurites) and in their associated white matter areas. The level of BI tau was similar in both AD and control brains. In contrast, A68 was present only in the AD brains, but it was confined to those brain regions with abundant NFTs, neuropil threads, and senile plaques. We view the reductions in BC tau in fornix and frontal white matter to be a consequence of the reductions in their associated grey matter regions i.e., hippocampus and frontal grey matter. Although there is no strict relationship between the reduction of BC tau and the level of A68 within an individual brain, the comparison of the AD group with the control group suggests that the grey matter of the affected regions may be the site for the conversion of BC tau into A68. Further, this process may occur rapidly or via pathways that do not involve BI tau since the levels of BI tau were similar in AD and control brains. Although the complete sequence of events leading to the transformation of tau into A68 and paired helical filaments remains to be elucidated, our data provide compelling evidence that A68 proteins are generated from tau-proteins in selected regions of the AD brain where neurofibrillary lesions comprised of paired helical filaments accumulate.     

Immunohistochemical characterization of a set of monoclonal antibodies to human neuron-specific enolase.

This paper describes the immunohistochemical staining properties of four monoclonal antibodies (MAbs) (CF, EB, AD, and KB) which had been previously shown to be specific for purified neuron-specific enolase (NSE) by a solid-phase radioimmunoassay. In this study, the authors immunostained a spectrum of normal and neoplastic neuronal, "neuroendocrine," and nonneuronal tissues fixed in formalin and embedded in paraffin. Positivity was generally restricted to normal neuronal structures and neuronal tumors, including adrenal neuroblastoma, ganglioneuroblastoma, olfactory neuroblastoma, pheochromocytoma, carotid body paraganglioma, duodenal gangliocytic paraganglioma, and teratoma with neuroepithelial components. Three staining patterns of the normal or neoplastic neuronal structures were observed: two MAbs (CF and EB) stained predominantly the nerve fibers (axoplasm); one (AD) stained predominantly the cell bodies (perikaryon); and one (KB) stained both the axoplasm and the perikaryon. "Neuroendocrine" tumors such as pulmonary small cell carcinoma, pancreatic islet cell tumor, thyroid medullary carcinoma, and carcinoid tumors from various locations showed a variable staining pattern. Tumor cells undergoing mitotic division were usually positive regardless of type. Normal structures other than neuronal or "neuroendocrine," including normal glial cells, were negative. The authors also studied a range of glial cell tumors with MAbs CF and AD as well as with Dako polyclonal antiserum to NSE. The results showed that CF stained the axonal fibers in the normal white matter surrounding these tumors; it did not stain the tumor cells or the perikarya of neurons in the surrounding normal gray matter. AD stained the glioma cells as well as the perikarya and dendrites of neurons in the surrounding normal gray matter; it did not stain the axonal fibers in the surrounding normal white matter. By contrast, the polyclonal antiserum stained all of these structures. The high degree of staining specificity of the MAbs should prove them to be valuable in immunohistochemical diagnosis of tumors as well as in further understanding the role of NSE in neuronal differentiation.     

A peptidyl–prolyl isomerase,FKBP12, accumulates in Alzheimer neurofibrillary tangles

We investigated a possible role in Alzheimer's disease (AD) for FKBP12, a peptidyl–prolyl cis–trans isomerase known to be important in protein assembly, folding and transportation by using Western blotting and microscopic analyses in postmortem brain tissues from elderly controls and the patients with AD. FKBP12 was enriched and localized to neuronal cell bodies and neurites in control brains. Intense immunoreactivity was found in large neurons such as pyramidal cells. Many FKBP12 positive granules were located in the cytoplasm and the proximal portion of dendrites and axons, and in the nuclei. By contrast, the expression of FKBP12 in AD brains was lower than in control brains. Furthermore, numerous intracellular neurofibrillary tangles (NFTs) were stained for FKBP12 in the hippocampal CA1 subfield, subiculum, entorhinal cortex and angular gyrus. Neuritic pathology such as neuropil threads and dystrophic neurites (DNs) within senile plaques (SPs) and some reactive astrocytes were also immunolabeled for FKBP12 in AD. Double immunofluorescence staining showed dual labeling of intracellular NFTs for FKBP12 and tau. Similar results were obtained in reactive astrocytes for the combination of FKBP12 and glial fibrillary acidic protein (GFAP). Labeling for FKBP12 was dense in axons stained for highly phosphorylated neurofilament protein. Thus our results suggest that FKBP12 may be involved in neuronal or astrocytic cytoskeletal organization and in the abnormal metabolism of tau protein in AD damaged neurons.     

Calcium phosphatase calcineurin influences tau metabolism

Alzheimer's disease (AD) is characterized by neuronal loss and the accumulation of β-amyloid plaques and neurofibrillary tangles in the brain. Cerebrospinal fluid (CSF) levels of β-amyloid and tau/phospho-tau181 (ptau181) are also associated with AD. We have previously demonstrated that a single nucleotide polymorphism in calcineurin is associated with CSF ptau181 levels and AD progression. In this study, we demonstrate that calcineurin protein levels are inversely correlated with dementia severity and Braak tangle stage in AD brains, and calcineurin activity is globally reduced in AD brains. We then sought to model the observed changes in CSF tau by measuring extracellular tau in cultured cells. SH-SY5Y cells treated with calcineurin inhibitors produced reduced calcineurin activity and a corresponding increase in extracellular ptau181. These findings are consistent with our observations in AD patients, who have elevated CSF ptau181 and reduced calcineurin activity in brain extracts. Thus, we have identified a gene that contributes to AD pathology and has functional consequences on tau metabolism in cultured cells.     

Nitration of tau protein is linked to neurodegeneration in tauopathies

Oxidative and nitrative injury is implicated in the pathogenesis of Alzheimer's disease (AD) and Down syndrome (DS), but no direct evidence links this type of injury to the formation of neurofibrillary tau lesions. To address this, we generated a monoclonal antibody (mAb), n847, which recognizes nitrated tau and alpha-synuclein. n847 detected nitrated tau in the insoluble fraction of AD, corticobasal degeneration (CBD), and Pick's disease (PiD) brains by Western blots. Immunohistochemistry (IHC) showed that n847 labeled neurons in the hippocampus and neocortex of AD and DS brains. Double-label immunofluorescence with n847 and an anti-tau antibody revealed partial co-localization of tau and n847 positive tangles, while n847 immunofluorescence and Thioflavin-S double-staining showed that a subset of n847-labeled neurons were Thioflavin-S-positive. In addition, immuno-electron microscopy revealed that tau-positive filaments in tangle-bearing neurons were also labeled by n847 and IHC of other tauopathies showed that some of glial and neuronal tau pathologies in CBD, progressive supranuclear palsy, PiD, and frontotemporal dementia with parkinsonism linked to chromosome 17 also were n847-positive. Finally, nitrated and Thioflavin-S-positive tau aggregates were generated in a oligodendrocytic cell line after treatment with peroxynitrite. Taken together, these findings imply that nitrative injury is directly linked to the formation of filamentous tau inclusions.     

Biochemical analysis of tau proteins in argyrophilic grain disease,Alzheimer's disease,and Pick's disease : a comparative study

Although argyrophilic grain disease is characterized histopathologically by tau-positive lesions known as argyrophilic grains located predominantly in limbic brain regions in the absence of other diagnostic neuropathologies, the biochemical correlates of argyrophilic grains in gray and white matter have not been reported. Thus, we analyzed insoluble (pathological) tau proteins in five argyrophilic grain disease brains in comparison with those seen in Alzheimer's disease and Pick's disease. Analyses of separately dissected gray and white matter samples from various cortical regions revealed that pathological tau in argyrophilic grain disease was confined primarily to mediotemporal neocortical gray and adjacent white matter, and also to the allocortex, amygdala, and hippocampus. The amounts of sarcosyl-insoluble tau in all five cases were substantially lower than in Alzheimer's disease and Pick's disease, but the amounts of sarcosyl-insoluble tau in white matter were higher or comparable to that detected in gray matter from the same region, which distinguishes argyrophilic grain disease from Alzheimer's disease. The banding patterns of tau isoforms in argyrophilic grain disease varied: in three cases they were similar to Alzheimer's disease, but in two other cases, 4 microtubule binding repeat (4R) tau predominated, which distinguishes argyrophilic grain disease from classical Pick's disease. The differences between these three diseases were re-enforced by the predominance of straight tau filaments from argyrophilic grain disease brains. Thus, we conclude that argyrophilic grain disease is a distinct tauopathy characterized by prominent accumulation of argyrophilic grains in limbic brain regions in association with the characteristic tau biochemical and ultrastructural profile reported here.     

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.     

Spatial and temporal distribution of intracellular free cholesterol in brains of a Niemann-Pick type C mouse model showing hyperphosphorylated tau protein. Implications for Alzheimer's disease

Niemann-Pick type C (NPC) disease is a fatal hereditary neurovisceral disorder with diagnostically relevant intracellular accumulation of cholesterol in non-brain tissue, for example the spleen and fibroblasts. In the brain, many ballooned neurons are seen. Using filipin microfluorodensitometry, significant accumulations of free cholesterol in specified neurons have been described in NPC patients. The present study demonstrates spatial and temporal accumulation of free cholesterol in the brains of homozygous NPC (-(npc)/-(npc)) mice, a widely acknowledged mouse model, and in primarily cultured neurons therefrom. Intraneuronal storage of free cholesterol was already prominent at a pre-clinical stage in various grey matter areas of the murine cerebral cortex. Hippocampal areas showed differential development of the pathological distribution of free cholesterol. The pyramidal cells in the CA3 sector of Ammon's horn were affected much earlier than in CA1. Some of the deeper cerebral nuclei were affected only slightly, even at the final stage. Neurons (E15-E17) cultured in a cholesterol-free medium also showed massive accumulation of intracellular free cholesterol. In addition, brains from the murine NPC model for Alzheimer's disease (AD)-like changes in the microtubule-associated protein tau were tested using the Gallyas silver technique and AT8-immunolabelling, since both human diseases are accompanied by intraneuronal tangles made up of tau protein aggregations. Although the analysis failed to show classical silver-stainable tangles of the AD type in the NPC mice, tau protein phosphorylated at epitopes considered to represent early stages of AD was found. This further strengthens the concept that an alteration in cholesterol metabolism may play an important role in AD. The NPC mouse model may thus serve as a tool to analyse the role of cholesterol in initial changes of tau that eventually lead to the formation of tangles in both NPC and AD.     

Modulation of tau phosphorylation by the kinase PKR: implications in Alzheimer's disease

Double-stranded RNA dependent kinase (PKR) is a pro-apoptotic kinase that controls protein translation. Previous studies revealed that activated PKR is increased in brains with Alzheimer's disease (AD). Glycogen Synthase Kinase Aβ (GSK-3β) is responsible for tau phosphorylation and controls several cellular functions also including apoptosis. The goal of this work was to determine if PKR could concurrently trigger GSK-3β activation, tau phosphorylation and apoptosis. In AD brains, both activated kinases co-localize with phosphorylated tau in neurons. In SH-SY5Y cell cultures, tunicamycin and Aβ(1-42) activate PKR, GSK-3β and induce tau phosphorylation and all these processes are attenuated by PKR inhibitors or PKR siRNA. Our results demonstrate that neuronal PKR co-localizes with GSK-3β and tau in AD brains and is able to modulate GSK-3β activation, tau phosphorylation and apoptosis in neuroblastoma cells exposed to tunicamycin or Aβ. PKR could represent a crucial signaling point relaying stress signals to neuronal pathways leading to cellular degeneration in AD.     

Immunocytochemical characterization of glial fibrillary tangles in Alzheimer's disease brain.

Neurofibrillary tangle is a major cytoskeletal pathology in Alzheimer's disease brains, and has been considered to develop exclusively in neuronal cells. We examined brains with Alzheimer's disease and observed argyrophilic fibrillary tangles not only in cortical neurons but also in subcortical glial cells in the frontal and temporal white matter. The tangles in glial cells were immunolabeled by antibodies against tau and ubiquitin, and double immunocytochemistry analyzed by confocal laser scanning microscopy demonstrated that the cytoplasms of tangle-bearing glia were labeled by antibodies against transferrin and 2'3'-cyclic nucleotide 3'-phosphohydrolase. Ultrastructurally, they were made up of bundles of straight filaments 16 nm in diameter and constricted filaments. These results indicate that fibrillary tangles resembling neurofibrillary tangles may develop in oligodendrocytes in brains with Alzheimer's disease and are distinguishable from glial cytoplasmic inclusions observed in multiple system atrophy brains. We referred to them as glial fibrillary tangles. Glial fibrillary tangles commonly occurred in this disease condition, and glial cells might be involved under the pathological processes similar to neuronal cells.     

Signature tau neuropathology in gray and white matter of corticobasal degeneration

Corticobasal degeneration (CBD) is an adult-onset progressive neurodegenerative disorder characterized by L-dopa-resistant rigidity, focal cortical deficits, and variable dementia. The neuropathological hallmark of CBD is the deposition of filamentous inclusions in neurons and glia composed of hyperphosphorylated tau with only four microtubule-binding repeats (4R-tau). To characterize the regional burden of tau pathology in CBD, we studied 12 brains with the neuropathological diagnosis of CBD using biochemical and histochemical techniques. Eleven brain regions were evaluated including gray and white matter from frontal, parietal, temporal, and occipital lobes and cerebellum as well as basal ganglia. Although the distribution of tau pathology was variable, neuropathological and biochemical data showed a similar burden of tau abnormalities in frontal, temporal, and parietal lobes and basal ganglia of both hemispheres. This included abundant, sarkosyl-insoluble 4R-tau in both gray and white matter of two or more of these cortical regions and basal ganglia, and to a lesser extent, cerebellar white matter. The insoluble tau pathology in gray and white matter showed overlapping but distinct phosphorylated epitopes suggesting cell-type and subcellular localization (ie, cell bodies versus cell processes)-specific differences in tau phosphorylation. In contrast, soluble tau was composed of normal 4R/3R-tau ratios indicating no gross abnormality in tau splicing. Thus, although clinically heterogeneous, CBD is a distinct lobar and basal ganglionic tauopathy with selective aggregation of 4R-tau.     

GSK-3 dependent phosphoepitopes recognized by PHF-1 and AT-8 antibodies are present in different tau isoforms

It is widely known that the tau protein that forms the aggregates found in tauopathies like Alzheimer’s disease (AD) is hyperphosphorylated. Many of the sites that are hyperphosphorylated in AD can also be found phosphorylated in non-pathological control brains, although to a lesser extend. Among the different kinases that are able to phosphorylate tau in these sites, GSK-3 has emerged as a key effector of AD pathogenesis in view of its interaction with many of the proteins involved in the ethiology of AD. In this work, we have tested if control samples show only a decrease in the amount of phosphorylated tau molecules, or if the phosphorylation at different sites occurs in different tau isoforms, whereas in the pathological situation a single tau isoform is modified simultaneously at the different sites. Our results indicate that the second possibility takes place and that the differences in the phosphorylation of different tau isoforms could be due to a different subcellular distribution of these different tau isoforms in a neuron.     

Expression of the small heat-shock protein alphaB-crystallin in tauopathies with glial pathology

Intracellular accumulations of filamentous material composed of tau proteins are defining features of sporadic and familial neurodegenerative disorders termed "tauopathies." In Alzheimer's disease, the most common tauopathy, tau pathology is predominantly localized within neurons; however, robust glial pathology occurs in other tauopathies. Although the pathogenesis of tauopathies remains primarily unknown, molecular chaperones such as heat-shock proteins (HSPs) are implicated in these tau disorders as well as other neurodegenerative diseases characterized by the accumulation of insoluble protein aggregates such as alpha-synuclein in Parkinson's disease and polyglutamine in Huntington's disease. We analyzed a variety of tauopathies with antibodies to a panel of HSPs to determine their role in the pathogenesis of these disorders. Although HSPs are not found in neuronal tau inclusions, we demonstrate increased expression of the small HSP alphaB-crystallin in glial inclusions of both sporadic and familial tauopathies. alphaB-crystallin was observed in a subset of astrocytic and oligodendrocytic tau inclusions as well as the neuropil thread pathology in cellular processes, but the co-expression of alphaB-crystallin with tau inclusions was relatively specific to tauopathies with extensive glial pathology. Thus, increased alphaB-crystallin expression in glial tau inclusions may represent a response by glia to the accumulation of misfolded or aggregated tau protein that is linked to the pathogenesis of the glial pathology and distinct from mechanisms underlying neuronal tau pathology in neurodegenerative disease.     

Tissue compartments in laminae II–V of rabbit visual cortex — three-dimensional arrangement, size and developmental changes

The neuropil in laminae II/III and IV of the mature rabbit visual cortex is subdivided into (a) dendrite bundles consisting of apical dendrites of pyramidal cells and associated axons and glial processes, (b) bundles of myelinated axons ascending vertically from the white matter up to lamina IV and (c) neuropil between bundles comprising mainly thin unmyelinated axons, small dendrites and associated glial processes. In this investigation the three-dimensional structure of these compartments was analysed. In addition, the volume fractions of the three neuropil compartments, perikarya and blood vessels in the different laminae and their quantitative changes from the late fetal period up to young adulthood and in a group of aged animals were determined. Serial l-μm epoxy sections were analysed. Dendrite bundles are more numerous and more intensively intertwined in lamina II/III than in lamina IV. At 28 days after conception the tissue in laminae II–V consists of approximately equal amounts, i.e. between 40 and 50%, of perikarya and neuropil. The volume fraction of blood vessels is about 4% and does not change much during development. During the first 16 days after birth the volume fraction of the neuropil increases to more than 70%, and conversely the volume fraction of nerve cells decreases to about 20%. Later, significant changes are seen only when the volume fractions of the three neuropil compartments are considered separately. The volume fraction of the neuropil between bundles increases throughout all laminae investigated, whereas the volume fraction of dendrite bundles is found to decrease. After 1 month, in lamina IV further increase of the neuropil between bundles is less marked, because here the bundles of myelinated axons become visible as an additional compartment. In young adult animals, the volume fractions of dendrite bundles are about 28% in the upper half, 16% in the lower half of lamina II/III and 7% in lamina IV. The neuropil between bundles comprises about 52% in the upper half, 65% in the lower half of lamina II/III and 62% in lamina IV. In lamina IV 14% is occupied by bundles of myelinated axons. In aged animals, the volume fraction of the neuropil between bundles decreases significantly in all laminae investigated. From previous ultrastructural studies, the extracellular space is known to be about 30% larger in the neuropil between bundles than in the dendrite bundles. Moreover, due to the prevalence of thin cell processes, the degree of tortuosity is larger in the neuropil between bundles than in the dendrite bundles. The present results together with these previous data are an indication of lamina-specific differences in the structure of the extracellular space. This may help to explain the electrical conductivity in the respective laminae of the cerebral cortex. The time course of postnatal changes of the neuropil compartments coincides with fundamental steps of structural and functional maturation of the rabbit visual cortex that are documented in the literature, and thus may be a valid parameter to investigate the degree of maturation or aging by morphological means.     

Proteasome degradation of brain cytosolic tau in Alzheimer's disease

The proteasomal degradation of cytosolic, phosphorylation-independent tau in human brains is potentially linked to the pathogenesis of neurofibrillary pathology in Alzheimer's disease (AD). Previous studies showed that the active 20S proteasome core degrades recombinant tau effectively, which prompted this study to determine if there was evidence of proteasomal degradation of tau in human brain with a range of neurofibrillary pathology. Cytosolic proteins from temporal cortex were isolated from 30,000xgsupernatants by resolving in size-exclusion chromatogra-phy for assay of tau and proteasomal subunits by Western blots. Levels of tau and proteasome subunits varied from case to case, with a significant inverse correlation between the levels of tau and 20S β-subunits, and between 70-kDa tau and 11S β-subunits, suggesting that tau is a proteasomal substrate. The inability to detect tau in western blots on cases without neurofibrillary pathology is consistent with the hypothesis that the proteasome is capable of degrading normal tau with an intact projection domain at the amino-terminal end; however, as proteasomal function becomes impaired during aging, tau clearance is impeded. Tau accumulates in progressively larger and more heterogeneous forms in brains with neurofibrillary pathology. Under normal conditions, non-proteasomal proteases are capable of digesting recombinant-tau from both the amino- and carboxyl-terminal ends toward the mid-section, but are lack of chaperon-like activity to unfold carboxyl-terminal truncated tau accumulated in AD. Our results support the hypothesis that failure of proteasomal and non-proteasomal proteolytic clearance mechanisms leads to tau accumulation and progressive neurofibrillary degeneration in AD.     

Cortical Alzheimer Type Pathology Does Not Influence tau Pathology in Progressive Supranuclear Palsy

Alzheimer disease (AD) is characterized by numerous senile plaques (SP) in addition to widespread neocortical neurofibrillary tangles (NFT). Some elderly have pathologic aging (PA), which is characterized by numerous SP composed of diffuse amyloid deposits with few or no NFT confined to the limbic lobe. Both AD and PA represent a range of Alzheimer type pathology (ATP). Some cases of progressive supranuclear palsy (PSP) have concurrent ATP, but the relationship between ATP and PSP has not been addressed. In this study, a consecutive series of PSP cases were divided into three groups according to the degree of concurrent ATP – pure PSP, PSP/PA and PSP/AD. Braak NFT stage was significantly greater in PSP/AD compared with both PSP/PA and PSP. Among the pathologic variables studied in middle frontal, superior temporal and motor cortices, there were no differences between PSP and PSP/PA except for SP. In PSP/AD, there was greater neuronal tau pathology (pretangles, NFT and neuropil threads) in middle frontal and superior temporal cortices, probably a reflection of ATP since there was no comparable increase in PSP-related glial tau pathology in these regions. The APOEɛ4 allele frequency was significantly higher in PSP/PA and PSP/AD than in PSP. These results strongly argue that ATP in PSP represents independent disease processes even when present in the same brain.     

Ubiquitin immunoreactive structures in normal human brains. Distribution and developmental aspects

Ubiquitin-immunoreactive structures in normal human brains ranging in age from 2 months to 91 years were studied with light and electron microscopy. Antibodies to ubiquitin immunostained structures in both neurons and glia. In the cerebrum, ubiquitin-immunoreactive, coarsely granular structures were most consistent with dystrophic neurites. They were most numerous in middle and upper cortical layers, especially lamina II of the entorhinal cortex and the cortical and accessory basal nuclei of the amygdala. Dystrophic neurites were first detected in brains of young adults, increased with age, and were numerous in the oldest brains. One of the normal elderly subjects had a small number of senile plaques with dystrophic neurites similar to those in the gray matter of the other brains, except for their location adjacent to amyloid deposits. With immunoelectron microscopy, dystrophic neurites were nonmyelinated neuronal processes containing dense, lamellar bodies, and finely granular material. White matter consistently had more immunoreactive structures than gray matter at all ages. The immunoreactive structures in white matter were smaller, less coarsely granular "dot-like" structures. With immunoelectron microscopy, dot-like structures were composed of dense inclusions within glial cells and focal swellings in myelin lamellae containing heterogeneous dense material. Only rarely were axons immunostained. Axonal spheroids in the basal ganglia, substantia nigra, and dorsal medulla were ubiquitin-immunoreactive. Spheroids were detected in these locations as early as the second decade, and they increased in number with age. A few dystrophic axons could be detected in spinal nerve roots of the oldest subjects. Other ubiquitin-immunoreactive structures included nuclei of small granular neurons, especially those in lamina II of the neocortex of the youngest brains; round cytoplasmic inclusions in tanycytes of all brains; and intranuclear Marinesco bodies in the substantia nigra and eosinophilic cytoplasmic inclusions in inferior olivary neurons in the oldest brains. These results demonstrate the spectrum of ubiquitinated structures in normal brains and suggest that progressive axonal dystrophy may be a more common age-related pathologic alteration of the brain than formerly recognized.     

Amyloid beta peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures

The neuropathological features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta protein (Abeta), intracellular neurofibrillary tangles containing phosphorylated tau protein and the loss of basal forebrain cholinergic neurons which innervate regions such as the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Abeta accumulation in vivo may initiate phosphorylation of tau protein, which by disrupting neuronal network may trigger the process of neurodegeneration observed in AD brains. However, the underlying cause of degeneration of the basal forebrain cholinergic neurons and their association, if any, to Abeta peptides or phosphorylated tau remains mostly unknown. In the present study, using rat primary septal cultures, we have shown that aggregated Abeta peptides, in a time (18-96 h)- and concentration (0.7-60 microM)-dependent manner, induce toxicity and decrease choline acetyltransferase enzyme activity in cultured neurons. Using immunocytochemistry and immunoblotting, we have also demonstrated that Abeta treatment can significantly increase the phosphorylation of tau protein in septal cultures. At the cellular level, hyperphosphorylated tau is mostly apparent in the somatodendritic compartment of the neurons. Abeta peptide (10 microM), in addition to tau phosphorylation, also activates mitogen-activated protein kinase and glycogen synthase kinase-3beta, the two kinases which are known to be involved in the formation of hyperphosphorylated tau in the AD brain. Exposure to specific inhibitors of the mitogen-activated protein kinase (i.e. PD98059) or glycogen synthase kinase-3beta (i.e. LiCl) attenuated the hyperphosphorylation of the tau protein in cultured neurons.Given the evidence that tau phosphorylation can induce cell loss by disrupting neuronal cytoskeleton, it is likely that aggregated Abeta peptide triggers degeneration of septal neurons, including those expressing the cholinergic phenotype, by phosphorylation of the tau protein activated by mitogen-activated protein kinase and glycogen synthase kinase-3beta. These results, taken together, suggest that cultured septal cholinergic neurons are vulnerable to Abeta-mediated toxicity and tau phosphorylation may play an important role in Abeta-induced neurodegeneration.